Strain and temperature dependent aggregation of Candida auris is attenuated by inhibition of surface amyloid proteins

Highlights • The amyloid inhibitor Thioflavin-T inhibited C. auris aggregation.• Aggregating isolates do not exhibit any defects in cell separation.• Genomic differences were identified between strongly aggregating and weakly-aggregating strains of C. auris.• Aggregation did not correlate with surface charge or hydrophobicity of yeast cells.


HIGHLIGHTS: 23
The amyloid inhibitor Thioflavin-T inhibited C. auris aggregation. 24 Aggregating isolates do not exhibit any defects in cell separation. 25 Genomic differences were identified between strongly aggregating and weakly-aggregating 26 strains of C. auris. 27 Aggregation did not correlate with surface charge or hydrophobicity of yeast cells. that may relate to the emergence of C. auris as an organism of major health care concern. In 60 this study, we focus on the ability of some strains to form clumps of aggregated yeast cells. that aggregating strains are attenuated in virulence as compared to weakly-aggregating 79 strains (Borman, Szekely, and Johnson 2016). This difference in virulence was also borne out 80 by a later study in a neutropenic murine model, although large aggregations of cells were 81 found in hearts, kidneys and liver of all mice suggesting that most strain will form aggregates 82 in tissue (Forgacs et al. 2020). Aggregating strains also have been shown to cause an 83 increased pro-inflammatory response on skin and at sites of wounds (Brown et al. 2020). 84 These studies provide evidence of the clinical importance of aggregation in C. auris and the 85 need to further dissect its aetiology. 86 87 Borman and colleagues reported that some C. auris daughter cells from aggregating 88 isolates may not be released normally from mother cells post cytokinesis (Borman,Szekely,89 and Johnson 2016). It was later observed that deletion of ACE2 transcription factor in a 90 weakly-aggregating C. auris strain was associated with decreased expression of chitinase 91 gene CTS1 that might participate in septal plate separation and hence aggregation (Santana 92 and O'Meara 2021). Deletion of ACE2 in Candida glabrata also resulted in clumping of cells 93 and in this fungus the aggregating mutant was hyper virulent (Kamran et al. 2004 Cell surface charge for the eight C. auris isolates was determined using the cationic 173 Alcian Blue dye as previously described (Hobson et al. 2004). Briefly, overnight grown cells 174 were harvested and washed twice with sterile PBS. They were suspended in 1 ml PBS and 175 adjusted to 1x10 7 cells/ml using Vi-Cell Blu cell counter. Aliquots of 1 ml were pelleted and 176 suspended in 1 ml 30 µg/ml Alcian Blue solution and incubated in the dark at room temperature 177 for 15 min. The aliquots were centrifuged and the supernatant was used to quantify Alcian 178 Blue concentration (free/un-bound) at 620nm. This was then used to determine concentration 179 of Alcian Blue dye bound to cells as described previously (Hobson et al. 2004). 180 181

Hydrophobicity assay 182
Cell surface hydrophobicity (CSH) was determined as described by (Rosenberg et al. 183 1996). Briefly, cells grown overnight were harvested and washed twice with sterile PBS. A cell 184 suspension at OD600 of 0.5 was prepared in 3 ml PBS (A0) and was overlaid with 0.4 ml 185 hydrophobic hydrocarbon, n-hexadecane (Sigma-Aldrich). After vigorous vortexing, the two 186 phases were allowed to separate for 10 min at 30°C. The optical density of aqueous phase 187 was measured (A1) and percentage CSH of the eight isolates was determined using following The cell wall of eight C. auris isolates was incubated with Fc-Pattern Recognition Receptors 208 fusion proteins (Fc-PRRs) to label exposed mannans (CRD4-7-Fc (Mannose Receptor), DC-209 SIGN and dectin-2), β-glucan (with PRR dectin-1) and exposed chitin (Wheat Germ  One-tenth volume of cDNA (2 µl) was added to qPCR reactions containing target specific 247 primers (Table 2)  The direction and magnitude of natural selection for each isolate were assessed by 299 measuring the rates of non-synonymous substitution (dN), synonymous substitution (dS) and 300 omega (ω = dN/dS) using the yn00 program of PAML (Yang 2007), which implements the 301 Yang and Nielsen method, taking into account codon bias (Yang and Nielsen 2000). The 302 program was run on every gene in each isolate using the standard nuclear code translation 303 We first confirmed and assessed the extent of aggregation variability in eight clinical 309 isolates of C. auris (Table 1), which is a property that is suspected to contribute to virulence 310 and immune evasion strategies (Borman, Szekely, and Johnson 2016). Four of the eight 311 isolates exhibited a strong aggregating phenotype (Fig. 1)  To explore the genetic differences between strongly-aggregating and weakly-325 aggregating isolates, we undertook an in silico screen of polymorphic genomic regions with 326 an emphasis on genes encoding secreted proteins. Depth of coverage plots indicated that 327 there were few obvious chromosomal copy number variation (CNV) (Fig. 2C) We identified 79 gene presence/absence polymorphisms (P/A polymorphisms; defined 333 as having breadth of coverage < 10% across the gene length) in one or more strains ( Fig. 2A,  334 B). None of the P/A polymorphisms were found exclusively in strongly aggregating isolates or 335 weakly aggregating isolates, and none of the isolate's genomes lacked essential genes 336 encoded by the Clade I reference genome involved in budding, cell division or separation, 337 suggesting that gene loss is not the cause of aggregation across these strains. 338

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Many of the P/A polymorphisms were due to deletions identified in several isolates. this weakly-aggregating isolate did not even form small aggregates of <4 cells. Given that only 358 68 of the 5,268 predicted protein encoding genes in C. auris had a high probability of encoding 359 a GPI anchor, this represents a significant enrichment among those gene deletions 360 (Hypergeometric test p-value = 5.51E -11 ). GO-term analysis of these P/A polymorphisms 361 revealed that 77.8% were associated with cell wall, membrane and extracellular proteins. 362 Together, this data suggests that loss of aggregation in isolate NCPF 13029 is likely caused 363 by the loss of one or more cell surface adhesins rather than a defect in cell separation. 364

365
To explore if signatures of positive selection underlie the aggregation phenotype, we 366 identified genes with signatures of positive selection (dN/dS > 1) compared with the Clade I 367 reference strain, identifying only 10 genes in total ( Fig. 2A). Again, most of these genes (n = 368 6/10) were in isolate NCPF 13029 and were mostly poorly characterised (all lacked GO-terms, 369 and none have previously been reported to be involved in aggregation). Therefore, this 370 approach has not identified any gene candidates for aggregation, although those genes may in the strongly-aggregating isolate NCPF 8977 with the weakly-aggregating isolate NCPF 379 13029. We also compared expression of chitin synthases (CHS1, CHS2, CHS3, CHS4 and 380 CHS8) that regulate septum formation leading to cell separation. We found that expression 381 levels of CTS1 were comparable between the strongly aggregating and weakly-aggregating 382 isolate. However, expression of CTS2 was substantially lower in the strongly aggregating 383 isolate compared to the weakly-aggregating isolate (Fig. 3A). Additionally, the strongly 384 aggregating isolate expressed substantially lower expression of CHS1 and CHS2 compared 385 to the weakly-aggregating isolate. The CHS3 mRNA level was low in the weakly-aggregating 386 isolate but higher in the aggregating isolate (Fig. 3B). Therefore, the transcript expression of 387 most genes involved in the formation and dissolution of the chitinous septum formation was 388 lower in the strongly-aggregating isolate than the weakly-aggregating isolate. 389 390

Cell separation in C. auris clinical isolates 391
We investigated if temporal differences in cell separation or defects in separation of 392 daughter cells from mother cells explained the aggregation phenotype by using a combination 393 of microscopy and microfluidics. Individual yeast cells from strongly aggregating and weakly-394 aggregating isolates were trapped in CellASIC microfluidic system and growth was assessed 395 for 8 h. Each isolate was grown in the presence of enriched YPD medium supplemented with 396 1 µg/ml Calcofluor White (CFW) to visualise cell wall chitin and the chitin rich septal wall 397 separating a mother and daughter cell. The architecture of septal rings and unipolar cell growth 398 of daughter cells emerging from mother cells was followed. Over a period of 8 h, normal chitin-399 rich septal rings were observed in all dividing C. auris isolates including aggregating strains 400 (Fig. 4) aggregating and weakly-aggregating isolates to polystyrene microspheres was determined. 415 We found that adhesion capacity of strongly aggregating isolates was comparable to the 416 weakly-aggregating isolates (Fig. 5A). The NCPF 8996 aggregate forming isolate had the 417 lowest adhesion capacity (65.5% of cells attached to microspheres) of the eight tested 418 isolates. Therefore, there was no correlation between adhesion capacity and aggregation 419 since high and low adhesion capacity was found in both strongly aggregating and weakly-420 aggregating strains. However, the adhesion capacity of C. auris isolates was substantially 421 greater (ranging from 65.5% to 94.4% cells attached to microspheres) than the C. albicans

Inhibition of amyloid protein function attenuates adhesion and aggregation 426
Many adhesins possess amyloid-forming sequences that can be hypothesised to make 427 a contribution the capacity for aggregation. Thioflavin-T binds and inhibits surface amyloids 428 and therefore may also inhibit aggregation. Therefore, C. auris isolates were incubated with 429 30 µM Thioflavin-T for 1 h and 24 h. We quantified surface amyloids in all isolates, and 430 assessed adhesion following inhibition of surface amyloids and quantified microscopically the 431 aggregation of isolates exposed to Thioflavin-T. We discovered that all aggregating isolates 432 had greater staining for surface amyloid proteins compared to weakly-aggregating isolates 433 (Fig. 5E). Following incubation with Thioflavin-T for 24 h, the capacity of these cells to adhere 434 to polystyrene microsphere was also reduced (Fig. 5B). As expected, adhesion of Thioflavin-435 T treated isolates to polystyrene microspheres was substantially lower than their untreated 436 counterparts. An average 2 fold decrease was observed for all tested C. auris isolates 437 following Thioflavin-T treatment (average % adherent cells = 85% before treatment and 34.4% 438 after treatment). Furthermore, the aggregating isolate NCPF 13052 showed a 3.47 fold 439 decrease (from 93.7% to 27.2%) in adhesion following Thioflavin-T treatment. However, 440 microscopy revealed that even after 24 h of incubation with Thioflavin-T, aggregating-isolates 441 continued to form aggregates (Fig. 5C, D). Aggregation was attenuated for strongly-442 aggregating isolates under the conditions tested, but was not completely suppressed. These 443 observations suggest that amyloid formation is important for aggregation but that other 444 unidentified factors may also contribute to aggregation. Alcian Blue binding would be indicative of a higher net negative charge on cell surface. As 464 seen with hydrophobicity, no link was found between the aggregation phenotype and surface 465 charge (Fig. 6B). Strongly-aggregating isolates, NCPF 13052 and NCPF 13059 exhibited the 466 highest Alcian Blue binding (0.38 pg/cell) and lowest binding (0.08 pg/cell) respectively. 467 isolates, its binding to the weakly-aggregating isolate NCPF 13029 was comparable to 481 strongly-aggregating strains. 482 The percent of principle carbohydrates (glucose, glucosamine and mannose) that 483 make up the cell wall polysaccharides was determined using High Pressure Ion 484 Chromatography (Fig. 7B). We discovered that despite each isolate having differential Fc-485 PRR probe binding, the percent of glucose, mannose and glucosamine in the cell wall of all 486 eight isolates were broadly comparable to each other and no statistically significant differences 487 were observed for any of these basic carbohydrates between strongly aggregating and 488 weakly-aggregating isolates. This experiment suggested that although isolate specific 489 differences were observed in the level and organization of cell wall exposed β-glucan, exposed 490 chitin and mannans, these differences are unlikely to explain the aggregation phenotype. 491 492

Effects of temperature on aggregation: 493
Three of the four weakly-aggregating isolates demonstrated some increased tendency 494 for aggregation when incubated at 37°C. This led us to investigate the effects of temperature 495 on aggregate formation in C. auris isolates. The eight isolates were pre-grown at 30°C and 496 further incubated at 30°C and 37°C for 24 h. All tested isolates displayed a substantial increase 497 in the number of aggregates formed at 37°C compared to 30°C (Fig. 8). NCPF 13029 and 498 NCPF 8985, were very weakly aggregating at 30°C also exhibited some degree of aggregation 499 at 37°C. protein(s) could contribute to aggregation in C. auris. Therefore, we determined expression of 510 these crucial genes involved in morphological changes and biofilm formation. Of the tested 511 target genes, expression of XOG1 was greater in strongly-aggregating isolate (NCPF 8977; 512 1.47x compared to weakly-aggregating isolate (NCPF 13029; 0.66) (Fig. 9). Other tested 513 target genes did not show a substantial difference in expression between the two isolates. 514 Furthermore, expression of XOG1 was substantially greater in the strongly-aggregating isolate 515 NCPF 8977 at 37°C (4.12X) compared to that at 30°C (1.47X). It is therefore possible that 516 expression differences of Xog1 β-exoglucanase contributes to aggregation. 517 518

Extracellular material production in aggregating isolates 519
We examined extracellular material (ECM) formation in the eight strains grown at 30°C 520 and 37°C. Small amounts of ECM were seen between adjacent cells of aggregating isolates 521 that had been grown at 30°C which displayed a rough cell surface (Fig. 10). The amount of 522 ECM observed on SEMs varied between aggregating isolates. The aggregating strains NCPF 523 13052 and NCFP 13059 appeared to be coated in a 'blanket-like' layer of ECM. Conversely, 524 aggregating strains NCPF 8977 and NCPF 8996 displayed ECM that was unevenly distributed 525 on the cell surface and was more concentrated where yeast cells adhered to each other. At 526 37°C, ECM was imaged in all eight isolates to varying degrees thus corroborating previous 527 observations of increased aggregation of "weakly-aggregating" isolates at higher temperature. 528 Based on these findings the production of ECM may also contribute to aggregation. McCreath, Specht, and Robbins 1995). Some differential levels of CHS expression were 558 observed. However, the high potential for post-transcriptional regulation of chitin synthases 559 limits the capacity to make conclusive inferences from these observations. We also did not 560 detect any deletions of core genes (According to Clade I reference genome gene annotation) 561 involved in cell division or separation in sequenced aggregating isolates. Importantly, time 562 lapse microscopy and microfluidics experiments did not reveal obvious defects in cell 563 separation in aggregating and weakly-aggregating strains. Therefore, our data suggests that 564 differences in the aggregation phenotype in our C. auris isolates is not governed by defects in 565 cell separation (our hypothesis 1). 566 567 A number of biophysical parameters were investigated in this study. Neither cell wall 568 charge nor cell surface hydrophobicity correlated with higher levels of aggregation, despite 569 the importance of both cell wall charge and hydrophobicity to the biology and virulence of 570 fungal pathogens (Gow and Lenardon 2023). We found that aggregation was enhanced at 571 deletions spanning entire genes (also known as P/A polymorphisms) in weakly-aggregating 599 isolate NCPF 13029 in GPI anchor proteins including Als4, which is a known adhesin in C. 600 albicans. Some of these proteins possess the amyloid-forming sequence that promotes 601 protein aggregation (Rauceo et al. 2004;Ramsook et al. 2010). More recently, certain C. auris 602 clinical isolates were found to exhibit Als4-mediated aggregation (Bing et al. 2023). We 603 therefore suspected that C. auris isolates that aggregate may express more amyloid-proteins 604 than weakly-aggregating isolates. This was confirmed by cell staining with Thioflavin-T, which 605 is an amyloid inhibitor. Prolonged incubation with Thioflavin-T resulted in a dramatic decline 606 in aggregation and adhesion in all isolates. Thioflavin-T did not inhibit aggregation completely 607 and it is therefore likely that aggregation might involve additional factors. For example, the 608 ZAP1 regulon has been shown to contribute to aggregation in C. albicans under zinc limitation 609 (Kumar et al. 2017). There may also be a combination of factors that collectively contributed 610 to aggregation that could differ in relative terms in different strains. In this study we have demonstrated that the aggregation phenotype is unlikely to be 626 caused by cell separation defects but may involve cell surface amyloid proteins and other 627 components of the cell wall and extracellular matrix. Our data suggests that aggregation is a 628 complex polygenic property of C. auris strains that also responds to key environmental 629 parameters such as environmental temperature. 630 631

Declaration of Competing Interest 632
The authors declare no financial interests/personal relationships which may be considered as 633 potential competing interests: 634

Ethical statement 635
No animals were used in this study that would be covered under Home Office legislation, UK